Probing the Bimolecular Interactions of Parathyroid Hormone and the Human Parathyroid Hormone/Parathyroid Hormone-Related Protein Receptor. 2. Cloning, Characterization, and Photoaffinity Labeling of the Recombinant Human Receptor

Parathyroid hormone (PTH) acts to regulate calcium homeostasis by interacting with a G-proteincoupled receptor that also binds PTH-related protein (PTHrP). In this report we describe the cloning, characterization, and biological activity of the cloned human (h) PTH/PTHrP receptor (Rc) and crosslinking of a benzophenone-substituted PTH analog, [Nle^8,18,Lys¹³(ε-pBz₂),L-2-Nal²³,Tyr³⁴]bPTH(1-34)-NH₂ (K13), to cells endogenously expressing the Rc and cells transiently or stably transfected with the human Rc. A full-length cDNA clone was isolated and fully sequenced from a human kidney cDNA library. Northern blot analysis of normal human tissues revealed a limited tissue distribution: a single transcript of ~2.3 kb was detected in kidney, lung, placenta, and liver. In human embryonic kidney cells (HEK-293, clone C-21) stably transfected with hPTH/PTHrP Rc, a single 85-90 kDa Rc-hormone complex was formed after photolysis in the presence of K13. This covalent cross-linking reaction was specifically inhibited by excess quantities of biologically active 1-34 analogs of bovine (b) PTH or hPTHrP but not by C-terminal and midregion PTH peptides. Photoincorporation of ¹²⁵I-labeled K13 into the Rc occurred with high efficiency (60-70%), approximately an order of magnitude greater than that achieved with conventional aryl azide cross-linking reagents. These results support the feasibility of our approach for specifically cross-linking a tagged PTH analog to the Rc, as a first step in the effort to identify directly the amino acid residues that constitute the Rc binding site.